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用皮脂刺激尾蚴,收集钻腺分泌物,获得粗制抗原,经层析聚焦获得纯化蛋白酶。用两种方案免疫动物。第一方案:小鼠腹腔注射尾蚴50条,取尾蚴分泌物与福氏完全佐剂混合,加强免疫3次后,取小鼠脾细胞与骨髓瘤细胞融合。第二方案为体外免疫方法:小鼠腹腔注射尾蚴100条,8周后取其脾细胞体外培养,将尾蚴分泌物加入脾细胞悬液,培养48小时后取脾细胞与骨髓瘤细胞进行融合。用ELISA方法检测杂交瘤细胞培养液中的抗体。将阳性孔内的细胞进一步克隆,然后保存在不含HAT的HB-101培养基内。用ELISA方法检测抗原。抗原包括尾蚴分泌物、卵分泌物、粗制成虫提取液及纯化尾蚴钻腺蛋白酶。标记抗体为β-半乳糖苷酶标记的兔抗鼠免疫球蛋白抗体。酶底物为O-苯酚-B-吡喃半乳糖苷。颜色反应分0～+++4个等级。此外,还检测单克隆抗体的亚
Sebum stimulation with cercariae, gonoporosis gland secretion collected crude antigen purified by chromatography to obtain protease. Animals were immunized with both regimens. The first program: mice injected cercariae 50 peritoneal cesarean section, take the secretions of cercariae and Freund’s complete adjuvant mixed to strengthen the immune 3 times, the mouse spleen cells and myeloma cells were fused. The second program is an in vitro immunization method: 100 mice were injected intraperitoneally with cercariae. After 8 weeks, the spleen cells were cultured in vitro, and the secretions of cercariae were added to the spleen cell suspension. After 48 hours of culture, the spleen cells and myeloma cells were fused. Antibodies in hybridoma cell culture media were detected by ELISA. Cells in the positive wells were further cloned and then stored in HAT-free HB-101 medium. Antigen was detected by ELISA. Antigens include cercariae secretions, egg secretions, crude adult extract and purified cercaria rhizobia. The labeled antibody is a beta-galactosidase labeled rabbit anti-mouse immunoglobulin antibody. The enzyme substrate is O-phenol-B-galactopyranoside. Color reaction points 0 ~ +++ 4 levels. In addition, monoclonal antibody subunits were also tested